Helicobacter ISSN 1523-5378

doi: 10.1111/hel.12217

Intrafamilial, Preferentially Mother-to-Child and Intraspousal,

Helicobacter pylori Infection in Japan Determined by Mutilocus
Sequence Typing and Random Amplified Polymorphic DNA
Fingerprinting
Shin-ichi Yokota,* Mutsuko Konno,† Shin-ichi Fujiwara,† Nariaki Toita,† Michiko Takahashi,†
Soh Yamamoto,* Noriko Ogasawara*,‡ and Tsukasa Shiraishi*
*Department of Microbiology, Sapporo Medical University School of Medicine, Sapporo, Japan, †Department of Pediatrics, Sapporo Kosei General
Hospital, Sapporo, Japan, ‡Department of Otolaryngology, Sapporo Medical University School of Medicine, Sapporo, Japan

Keywords
Intrafamilial infection, intraspousal infection,
Helicobacter pylori, multilocus sequence
typing, random amplified polymorphic DNA
fingerprinting.
Reprint requests to: Mutsuko Konno, Department
of Pediatrics, Sapporo Kosei General Hospital,
North-3, East-8, Chuo-ku, Sapporo 060-0033,
Japan. E-mail: mutsukon@ja-hokkaidoukouseiren.
or.jp
Abstract
Background: The infection route of Helicobacter pylori has been recognized to
be mainly intrafamilial, preferentially mother-to-child, especially in devel-
oped countries. To determine the transmission route, we examined whether
multilocus sequence typing (MLST) was useful for analysis of intrafamilial
infection. The possibility of intraspousal infection was also evaluated.
Materials and Methods: Clonal relationships between strains derived from
35 index Japanese pediatric patients, and their family members were ana-
lyzed by two genetic typing procedures, MLST and random amplified poly-
morphic DNA (RAPD) fingerprinting.
Results: Mostly coincident results were obtained by MLST and RAPD. By
MLST, the allele of loci in the isolates mostly matched between the index
child and both the father and mother for 9 (25.7%) of the 35 patients,
between the index child and the mother for 25 (60.0%) of the 35 patients.
Conclusions: MLST is useful for analyzing the infection route of H. pylori as
a highly reproducible method. Intrafamilial, especially mother-to-children
and sibling, infection is the dominant transmission route. Intraspousal infec-
tion is also thought to occur in about a quarter in the Japanese families.

Helicobacter pylori is an etiological agent of the develop-
ment of numerous gastrointestinal disorders, including
gastritis, gastric and duodenal ulcers, gastric adenocarci-
noma, and gastric lymphoma [1–3]. H. pylori is believed
to be acquired mostly under 5 years of age, because the
secretion of gastric acid is immature [4–8]. H. pylori
infection subsequently endures through most of the
host’s life unless being eradicated by chemotherapy.
Infection routes of H. pylori are considered to be fecal–
oral, gastro-oral and oral–oral, and socioeconomic sta-
tus and hygiene status are thought to be important for
the infection source [9,10]. In developing countries, a
horizontal transmission route, such as through contam-
inated water and food, and via intensive contact
between infants and nonparental care workers, who
commonly care for children from multiple families,
may concomitantly play a more important role than
intrafamilial infection [9,11]. In developed countries,
improved sanitization and differences in quality of life,
including childcare, compared to developing countries
have markedly reduced the risk of horizontal transmis-
sion. Intrafamilial infection has become therefore pref-
erential [12–14]. It has been proved that mother-to-
child(ren) infection is dominant in developed countries
[8,15].
For examination of the infection route, we and sev-
eral researchers use random amplified polymorphic
DNA (RAPD) fingerprinting, which is an easy and rapid
procedure [8,14–17]. However, this PCR-based finger-
printing pattern has low reproducibility among different
experiments and is dependent on detail experimental
conditions. Simultaneous comparison of specimens in
the same experiment is therefore necessary. Further-
more, similar banding patterns are occasionally
observed in strains originating from independent
clones, and determination sometimes becomes difficult.

© 2015 John Wiley & Sons Ltd, Helicobacter 1

Intraspousal Infection of H. pylori Yokota et al.

Recently, multilocus sequence typing (MLST), which Isolation and Culture of H. pylori
consists of the combination of partial nucleotide
Isolation and identification of H. pylori strains from the
sequences of several housekeeping genes, has been
biopsy specimens and gastric juice were performed as
used for clone discrimination and spread of specific
previously described [15,28]. The H. pylori isolates were
clones of various bacteria [18,19]. The databases are
cultured on Helicobacter-selection agar plates (Nissui
publicly available on web sites, and they can be easily
Pharmaceuticals, Tokyo, Japan) at 37 °C in a microaer-
accessed. In H. pylori, partial sequences of seven house-
ophilic atmosphere using AnaeroPack MicroAero (Mits-
keeping genes (atpA, efp, mutY, ppa, trpC, ureI, and
ubishi Gas Chemical, Tokyo, Japan). Consequently, 35
yphC) are used for the typing. Recently, MLST for
strains from the indexed patients, 26 strains from
H. pylori has been used for examination of the geo-
fathers, 28 strains from mothers, and 11 strains from
graphic origin and migration of H. pylori in the world
siblings were examined in this study.
[20–26]. Furthermore, Osaki et al. tried to apply MLST
for examination of the infection route using fecal speci-
mens [27]. In this study, we assessed MLST for investi- Purification of Genomic DNA
gation of intrafamilial infection using H. pylori isolates.
Genomic DNA, which was used as the template for
PCR, was isolated from bacterial cells using the Quick-
Methods Gene DNA tissue kit S (Fuji Film, Tokyo, Japan) and
the QuickGene 800 system (Fuji Film).
Subjects
The 35 index pediatric patients in this study were diag- RAPD Fingerprinting
nosed by gastrointestinal endoscopy and pathology as
RAPD fingerprinting analysis was performed as
having H. pylori gastritis with or without duodenal/gas-
described previously [15,28]. Briefly, the PCR was car-
tric ulcer disease. Their family members were recruited
ried out using 20 ng template DNA, 20 pmol primer,
for this study and underwent the H. pylori stool antigen
and HotStarTaq master mix (Qiagen, Hilden, Germany).
test (Premier Platinum HpSA PLUS, Meridian Biosci-
The PCR primers were selected from random primers of
ence, Cincinnati, OH, USA). The family members with
DNA Oligomer set A-4 (NIPPON GENE, Tokyo, Japan).
positive results of the HpSA test were invited to
Of the 12 primers (A01 to A12), the primers which had
undergo gastroscopy. All subjects were Japanese. Con-
given sufficient numbers and variable sizes of PCR
sequently, the 35 indexed patients, 26 fathers and 28
product bands in the preliminary study were selected.
mothers underwent gastroscopy and endoscopic biop-
Four primers (A04 (50 -ATCAGCGCACCA-30 ), A07 (50 -
sies. In addition, gastric juice samples were aspirated
TGCCTCGCACCA-30 ), A08 (50 -GCCCCGTTAGCA-30 ),
through a sterile nasogastric tube from 11 siblings
and A11 (50 -GATGGATTTGGG-30 )) were suitable for
under 15 years of age whose HpSA test was positive.
this study [8,15]. The cycling program was 35 cycles of
The patients and their family members had not received
94 °C for 2 minutes, 38 °C for 2 minutes, and 72 °C
antibiotics, proton-pump inhibitors, or nonsteroidal
for 2 minutes, followed by a final incubation at 72 °C
anti-inflammatory drugs within 1 month before the
for 10 minutes. The products were analyzed by 1.5%
specimens were taken. Informed consent was obtained
agarose gel electrophoresis. A 100-bp DNA ladder
from all patients and their family members and from
(Dooh Rika Sangyo, Sapporo, Japan) was used as a size
nursery teachers. This work was approved by the
marker. When RAPD patterns obtained from at least
Review Board of Sapporo Kosei General Hospital.
three primers were identical, it is judged that the strains
Biopsy specimens were taken using a sterilized
originated from the same clone.
endoscope from the gastric antrum and body for
H. pylori culture. The biopsy forceps were disinfected by
immersion in 0.05% phtharal for 5 minutes and then MLST
rinsed with water for each specimen collection. In one
MLST was determined according to the web site on
family (No. 35), the RAPD patterns and MLST of the
http://pubmlst.org/helicobacter/. Partial nucleotide
index patient and his two siblings were different from
sequences of eight housekeeping genes, atpA, efp, mutY,
those of their parents. As those three children had
ppa, trpC, ureI, yphC, and vacA, were determined by
attended the same nursery school, we studied H. pylori
the PCR direct sequence method. PCR was performed
strains isolated from gastric juice from two nursery
with HotStarTaq master mix. Primer sets for the eight
teachers who worked at the nursery school and were
genes were according to the H. pylori MLST Web site as
unrelated to the family.

2 © 2015 John Wiley & Sons Ltd, Helicobacter

Yokota et al. Intraspousal Infection of H. pylori

Table 2 Clonal analysis of strains isolated from the index patients and family members (Family No. 35), and nursery school teachers by MLST and
RAPD

Allele sequence type

Family membera H. pylori infectionb RAPD patternc atpA efp mutY ppa trpC ureI yphC vacA MLST No.

F + ● 2366 2220 2469 1125 2523 2525 2495 562 2824

M + ▲ 2315 2184 2351 445 2426 1968 2394 563 2761
C1 + ○ 951 909 950 2230 2427 2392 2400 564 2762
C2d + ○ 951 909 950 2230 2427 2392 2400 564 2762
C3 + ○ 951 909 950 2230 2427 2392 2400 564 2762
T1 + M 2395 2220 2439 2305 2524 2547 2461 525 2845
T2 + ■ 425 458 2467 420 2525 2477 2462 534 2847

a
RAPD analysis of this family was performed in this study. F: father, M: mother; C1: first born child, C2: second born child, C3: third born child,
and T1 and T2: teachers, who work in the nursery school where the children attended.
b
+: positive, : negative.
c
The same or closely similar RAPD patterns are denoted as the same symbol. Data are shown in Fig. 1.
d
Index patient.

transmission between father and child(ren) seems to be
rare, because mother-to-child infection is significantly
dominant compared to father-to-child infection, as
found in our previous study [15] and the present study.
According to statistics published by the Japanese Minis-
try of Health, Labor and Welfare, the time that the
father spends taking care of children (0.33 hour per
day) is much less than that of the mother (3.09 hour
per day) in Japan [39]. This indicates that physical close
contact of the child with the father is much less than
that with the mother. So the opportunity for transmis-
sion of H. pylori from child-to-father is considered to be
much less frequent than that for transmission between
spouses.
To our knowledge, there have only been a few stud-
ies in which the frequency of intraspousal infection
was investigated, and the results of those studies are
remarkably different. Georgopoulos et al. reported in-
traspousal infection frequency of 44% (8/18) in Greece
determined by ribopatterns [40]. Kivi et al. reported a
frequency of 21.7% (5/23) in Sweden determined by
RAPD and restriction fragment length polymorphism
(RFLP) [16]. Lee et al. reported a frequency of 46.7%
(7/15) in Western Australia determined by amplified

Figure 1 RAPD finger printing patterns of the strains isolated from the index patient and family members (Family No. 35), and two nursery school
teachers. PCR reaction was performed using DNA derived from each strain as a template and a primer (A04, A07, A08 or A11). The PCR products
were separated with 1.5% agarose electrophoresis and stained with ethidium bromide. S: DNA size marker, F: father, M: mother; C1: first born
child, C2: second born child (the index patient), C3: third born child, and T1 and T2: nursery school teachers.

© 2015 John Wiley & Sons Ltd, Helicobacter 7

Intraspousal Infection of H. pylori Yokota et al.

Table 1 Clonal analysis of strains isolated from the index patients and family members in 34 families by MLST and RAPD

Allele sequence type

Family No. Family membera H. pylori infectionb RAPD patternc atpA efp mutY ppa trpC ureI yphC vacA MLST No.

1d F + ○ 2322 2203 2362 56 2434 2409 2408 521 2766

M + ○ 2322 22041 2362 56 2434 2409 2408 521 2783
C1e + ○ 2322 2203 2362 56 2434 2409 2408 521 2766
C2 + ○ 2322 2203 2362 56 2434 2409 2408 521 2766
C3 + ○ 2322 2203 2362 56 2434 2409 2408 521 2766
2 F + ○ 1232 2206 2368 52 2436 2411 2410 522 2784
M + ○ 1232 2206 2368 52 2436 2411 2410 522 2784
C1 + ○ 1232 2206 2368 52 2436 2411 2410 522 2784
C2e + ○ 1232 2206 2368 52 2436 2411 2410 522 2784
3 F + ○ 2328 2219 2370 2248 2441 2425 2419 526 2787
M + ○ 2328 2219 23761 2248 2441 2425 2419 526 2788
C1e + ○ 2328 2219 2370 2248 2441 2425 2419 526 2787
C2 + ○ 2328 2219 2370 2248 2441 2425 2419 526 2787
4d F + ○ 2319 2191 23901 945 954 36 957 529 2790
M + ○ 2319 2191 2355 945 954 36 957 529 2757
C1e + ○ 2319 2191 2355 945 954 36 957 529 2757
C2 + ○ 2319 2191 2355 945 954 36 957 529 2757
5 F + ○ 2331 2220 2384 1125 2442 2426 2422 527 2789
M + ○ 2331 2220 2384 1125 2442 2426 2422 527 2789
C1e + ○ 2331 2220 2384 1125 2442 2426 2422 527 2789
C2 + n.d.
6 F + ○ 2327 2213 1865 2246 2440 2419 2417 524 2786
M + ○ 2327 2213 1865 2246 2440 2419 2417 524 2786
C1 + n.d.
C2e + ○ 2327 2213 1865 2246 2440 2419 2417 524 2786
7d F + ○ 2360 2246 2425 502 2499 2471 2453 542 2802
M + ○ 2360 2246 2425 502 2499 2471 2453 5432 2802
C1e + ○ 2360 2246 2425 502 2499 2471 2453 542 2802
C2 
8d F + ○ 1332 2212 2369 2244 2438 2418 2414 523 2785
M + ○ 1332 2212 2369 2244 2438 2418 2414 523 2785
C1e + ○ 1332 2212 2369 2244 2438 2418 2414 523 2785
9d F + ○ 1760 2185 2354 2232 457 2393 457 528 2754
M + ○ 1760 2185 2354 2232 457 2393 457 528 2754
C1e + ○ 1760 2185 2354 2232 457 24001 457 528 2755
10d F + ● 2305 2266 2342 2153 2502 2390 2307 544 2803
M + ○ 2366 2269 2347 1125 2419 2391 2345 460 2804
C1e + ○ 23091 2269 2347 1125 2419 2391 2345 54610 2805
C2 + n.d.
C3 + n.d.
11 F + ● 2339 2234 2395 2256 2447 2445 2434 532 2793
M + ○ 2340 2236 2397 2257 2448 2553 2435 533 2794
C1e + ○ 2340 2236 2397 2257 2448 2553 2435 533 2794
C2 
C3 
12 F + ● 2356 2220 2414 2271 2475 2462 2448 538 2798
M + ○ 2357 2242 2419 445 2482 2463 2451 539 2799
C1e + ○ 2357 2242 2419 445 2482 2463 2451 539 2799
C2 
C3 

(Continued)

4 © 2015 John Wiley & Sons Ltd, Helicobacter

Yokota et al. Intraspousal Infection of H. pylori

Table 1 (Continued)

Allele sequence type

Family No. Family membera H. pylori infectionb RAPD patternc atpA efp mutY ppa trpC ureI yphC vacA MLST No.

13 F + ● 2313 2276 2359 942 2508 1968 2401 547 2806

M + ○ 2374 2202 2360 2235 2433 2406 2404 548 2807
C1 + ○ 2374 2202 2360 2235 2433 2406 2404 548 2807
C2e + ○ 2374 2202 2360 2235 2433 2406 2404 548 2807
14 F + ● 2377 2285 459 2282 458 1968 2463 549 2808
M + ○ 2382 2290 2368 2290 2513 2480 2469 550 2809
C1e + ○ 2382 2290 2368 2290 4571 2480 2469 550 2810
C2 
15 F + ● 255 2230 2393 226 2443 2442 2426 530 2791
M + ○ 2338 2232 2394 179 2445 2443 2431 531 2792
C1e + ○ 2338 2232 2394 179 2445 2443 2431 531 2792
16d F + ● 949 2244 946 2272 2491 969 460 540 2800
M + ○ 2313 462 2369 2274 2498 2470 2452 540 2801
C1e + ○ 2313 462 2369 2274 2498 2470 2452 540 2801
17 F + ● 2345 455 2399 2261 2419 966 1247 535 2795
M + ○ 2350 2237 2400 2266 1225 2456 2440 536 2796
C1e + ○ 2350 2237 2400 2266 1225 2456 2440 536 2796
C2 + ■ 2353 2239 2402 2268 2472 2460 2414 537 2797
18 F 
M + ○ 1760 455 2463 2295 2516 2506 2484 556 2817
C1 
C2e + ○ 1760 455 2463 2295 2516 2506 2484 556 2817
C3 
19 F 
M + ○ 2388 2308 2464 2296 2519 2507 2485 557 2818
C1e + ○ 2308 23091 2464 2296 2519 2507 2485 557 2819
C2 
C3 
20 F 
M + ○ 2364 2244 454 2276 458 2437 2447 536 2843
C1e + ○ 2364 2244 454 2276 458 2437 2447 536 2843
C2 
21 F 
M + ○ 940 1772 2451 2292 2516 2493 2481 553 2813
C1e + ○ 940 1772 2451 2292 2516 24951 2481 553 2814
22 F + ○ 2393 2313 2419 2298 252 2524 2493 561 2823
M + ● 2391 2312 2467 420 423 2516 2492 560 2822
C1 + n.d.
C2 
C3e + ○ 2393 2313 2419 2298 252 2524 2493 561 2823
23d F + ● 2340 2236 2397 2330 2448 2553 2435 577 2837
M 
C1 
C2e + ○ 2463 2327 2484 26 2538 1362 2551 578 2838
C3 
24 F + ● 2421 2320 2475 2306 2530 2536 2538 569 2829
M 
C1 + ○ 2430 453 2476 445 2531 2545 460 536 2830
C2e + ○ 2430 453 2476 445 2531 2545 460 536 2830
25d F + ● 1760 2330 449 445 2541 44 1937 581 2841
M 
C1e + ○ 2466 2331 2463 2337 2542 50 2557 582 2842
C2 + ○ 2466 2331 2463 2337 2542 50 2557 582 2842

(Continued)

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Intraspousal Infection of H. pylori Yokota et al.

Table 1 (Continued)

Allele sequence type

Family No. Family membera H. pylori infectionb RAPD patternc atpA efp mutY ppa trpC ureI yphC vacA MLST No.

26 F + ● 2439 2321 2477 2307 2532 2547 2539 571 2831

M 
C1e + ○ 2447 2322 2478 942 458 2548 2541 572 2832
27 F + ● 2458 2324 2481 420 2535 2551 1962 571 2835
M 
C1e + ○ 2461 2184 2482 45 2532 2552 2547 576 2836
28 F + ● 2449 455 2479 2308 2533 2549 2543 573 2833
M 
C1e + ○ 2452 2293 2480 2328 2534 2550 2546 574 2834
29d F + ● 2464 2328 2485 2331 2539 2554 2555 579 2839
M 
C1e + ○ 2464 2329 2486 2332 2540 2555 2556 580 2840
30d F 
M + ● 2386 909 2452 2294 2517 2504 2307 554 2815
C1e + ○ 1760 2307 2454 445 457 1247 457 555 2816
31 F 
M + ● 2389 2310 2465 2297 2520 2510 2486 558 2820
C1e + ○ 2390 2311 2466 502 2521 2514 2488 559 2821
32 F 
M + ● 2398 2316 2471 2301 2472 2529 2525 565 2825
C1e + ○ 2400 2317 2472 2301 2527 2533 2533 566 2826
33d F 
M + ● 940 2246 2450 502 2418 2482 2477 551 2811
C1e + ○ 2384 2298 2450 502 2515 2485 2477 552 2812
34 F 
M + ● 2357 2202 2360 2235 2433 2406 2535 548 2827
C1e + ○ 2420 2319 2474 1254 2529 459 2536 568 2828

Superscript numbers of right side are different numbers of nucleotides from the allele sequence of the same clone-derived strain(s) from other
family member(s).
a
F: father, M: mother; C1: first born child, C2: second born child, C3: third born child.
b
+: positive, : negative.
c
The same or closely similar RAPD patterns in the family are denoted as the same symbol. n.d.: H. pylori infection is positive, however, isolation
of H. pylori strains has not been performed.
d
RAPD analysis of the family was performed in this study. These are not included in the previous study [8,15].
e
Index patient.

sequence type numbers are next to the previously reg-
istered one. Information on similarities and relation-
ships between the sequences is therefore not included
in the type numbers. In such cases, comparison of
nucleotide sequences should be required.
We previously found that mother-to-child infection
is the predominant route of intrafamilial clustering of
H. pylori in Japan by RAPD fingerprinting [8,15]. In the
present study, 21 (60.0%) of the 35 index patients
showed the same clone-derived strains of the mother
by MLST, which confirmed mother-to-child transmis-
sion as the major causative mode of infection. In this
study, we found that alleles of all of the seven loci in
H. pylori isolates matched those of all of the family
members in 9 of 35 (25.7%) by MLST, suggesting that
mother-to-child infection and intraspousal infection,
namely adult-to-adult, have occurred. There is a gen-
eral consensus in the literature that H. pylori infection
is usually acquired in early childhood [4–8] and persists
for life, and the frequency and importance of acquisi-
tion of H. pylori infection in adults are therefore consid-
ered to be low and underestimated. However, there
have been some studies on H. pylori infection in adults,
including studies on experimental infection [31,32],
acute gastric mucosal lesions [33,34], reinfection after
eradication [35–37], and infection via gastrointestinal
endoscopy [38].
The possibility that the same clone-derived strains in
spouses have been caused by child-to-father transmis-
sion cannot be completely ruled out. However, the

6 © 2015 John Wiley & Sons Ltd, Helicobacter

Yokota et al. Intraspousal Infection of H. pylori

Table 2 Clonal analysis of strains isolated from the index patients and family members (Family No. 35), and nursery school teachers by MLST and
RAPD

Allele sequence type

Family membera H. pylori infectionb RAPD patternc atpA efp mutY ppa trpC ureI yphC vacA MLST No.

F + ● 2366 2220 2469 1125 2523 2525 2495 562 2824

M + ▲ 2315 2184 2351 445 2426 1968 2394 563 2761
C1 + ○ 951 909 950 2230 2427 2392 2400 564 2762
C2d + ○ 951 909 950 2230 2427 2392 2400 564 2762
C3 + ○ 951 909 950 2230 2427 2392 2400 564 2762
T1 + M 2395 2220 2439 2305 2524 2547 2461 525 2845
T2 + ■ 425 458 2467 420 2525 2477 2462 534 2847

a
RAPD analysis of this family was performed in this study. F: father, M: mother; C1: first born child, C2: second born child, C3: third born child,
and T1 and T2: teachers, who work in the nursery school where the children attended.
b
+: positive, : negative.
c
The same or closely similar RAPD patterns are denoted as the same symbol. Data are shown in Fig. 1.
d
Index patient.

transmission between father and child(ren) seems to be
rare, because mother-to-child infection is significantly
dominant compared to father-to-child infection, as
found in our previous study [15] and the present study.
According to statistics published by the Japanese Minis-
try of Health, Labor and Welfare, the time that the
father spends taking care of children (0.33 hour per
day) is much less than that of the mother (3.09 hour
per day) in Japan [39]. This indicates that physical close
contact of the child with the father is much less than
that with the mother. So the opportunity for transmis-
sion of H. pylori from child-to-father is considered to be
much less frequent than that for transmission between
spouses.
To our knowledge, there have only been a few stud-
ies in which the frequency of intraspousal infection
was investigated, and the results of those studies are
remarkably different. Georgopoulos et al. reported in-
traspousal infection frequency of 44% (8/18) in Greece
determined by ribopatterns [40]. Kivi et al. reported a
frequency of 21.7% (5/23) in Sweden determined by
RAPD and restriction fragment length polymorphism
(RFLP) [16]. Lee et al. reported a frequency of 46.7%
(7/15) in Western Australia determined by amplified

Figure 1 RAPD finger printing patterns of the strains isolated from the index patient and family members (Family No. 35), and two nursery school
teachers. PCR reaction was performed using DNA derived from each strain as a template and a primer (A04, A07, A08 or A11). The PCR products
were separated with 1.5% agarose electrophoresis and stained with ethidium bromide. S: DNA size marker, F: father, M: mother; C1: first born
child, C2: second born child (the index patient), C3: third born child, and T1 and T2: nursery school teachers.

© 2015 John Wiley & Sons Ltd, Helicobacter 7

Intraspousal Infection of H. pylori
fragment length polymorphism (AFLP) and RAPD [41].
In the present study, it was shown that 9 (25.7%) of
the 35 couples were infected with the same clone-
derived H. pylori strains. These studies suggest that in-
traspousal transmission of H. pylori between adults
may occur with significant frequency. On the other
hand, Suzuki et al. reported that intraspousal transmis-
sion occurred in only one of 21 adults in Japan as
determined by RFLP [42]. Kuo et al. reported a fre-
quency of 1 of 25 in Taiwan as determined by RAPD,
RFLP, and DNA sequences of the vacA mid region [17].
Luman et al. reported a frequency of 0/13 in Singapore
as determined by RFLP [43]. The remarkable differ-
ences of frequency could be influenced by various fac-
tors, such as socioeconomic status, hygiene status, life
style, and age distribution, of the study population. In
our present study, we confirmed that intraspousal
infection occurred in about a quarter in Japan. Further-
more, strains derived from children of the nine families
were shown to be originated from the same clone.
Recently, Linz et al. examined the whole genome
sequences of a pair of strains from a couple are found
that they were almost identical using MLST, RAPD, and
AFLP analyses [29]. Genetic diversities, namely SNPs
and clusters of nucleotide polymorphisms, which are
thought to be imports of DNA fragments from other
strains, were observed between the two strains. These
genetic alterations and diversities may occur in infect-
ing H. pylori strains during host adaptation [29,30].
According to the report, whole genome analysis may
provide more information on the direction of bacterial
transmission in a family, including transmission from
one spouse to the other.
The infection rate of H. pylori is lower in developed
countries than in developing countries [44,45].
Recently, the infection rate in young people in Japan
has been decreasing [46]. It will become rare for both
members of a couple to be positive for H. pylori. So, in-
traspousal infection might increasingly become a con-
cern. Prospective studies are needed to prove
intraspousal infection, but such a study would be ethi-
cally difficult.
In 13 families, strains derived from children were
different from strains from both the mother and father
or different from the strain from one of the parents
with the other parent being H. pylori negative. The
source of infection in these children remains unclear.
In one family (No. 35, Table 2), strains from three sib-
lings were suggested to be originated from the same
clone; however, strains from the father and mother
were different from those of the children. In addition,
strains derived from two teachers of the nursery school
were different from each other and distinct from any
8 © 2015 John Wiley & Sons Ltd, Helicobacter
Yokota et al.
strains derived from the members of family No. 35. The
origin of infection in these index children has not been
clear.
In conclusion, MLST is useful for discrimination of
strains, for example, analysis of the infection route of
H. pylori, as a highly reproducible procedure. Intrafa-
milial, especially mother-to-children and sibling, infec-
tion is dominant, and intraspousal infection is thought
to have occurred in about a quarter of the subjects in
this study.
Acknowledgements and Disclosures
The authors thank Itaru Hosaka, Kohtaroh Akita, Natsumi
Matsumoto, and Saori Hashimoto for technical assistance.
Competing interests: All the authors declare that there is no
conflict of interests.
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